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14 juin 19331933/06/14 (A34,N11869)

Thiopental inhibits global protein synthesis by repression of eukaryotic elongation factor 2 and protects from hypoxic neuronal cell death.

Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited

Fluvoxamine for the treatment of COVID-19

Background Fluvoxamine is a selective serotonin reuptake inhibitor (SSRI) that has been approved for the treatment of depression, obsessive compulsive disorder, and a variety of anxiety disorders; it is available as an oral preparation. Fluvoxamine has not been approved for the treatment of infections, but has been used in the early treatment of people with mild to moderate COVID-19. As there are only a few effective therapies for people with COVID-19 in the community, a thorough understanding of the current evidence regarding the efficacy and safety of fluvoxamine as an anti-inflammatory and possible anti-viral treatment for COVID-19, based on randomised controlled trials (RCTs), is needed. Objectives To assess the efficacy and safety of fluvoxamine in addition to standard care, compared to standard care (alone or with placebo), or any other active pharmacological comparator with proven efficacy for the treatment of COVID-19 outpatients and inpatients. Search methods We searched the Cochrane COVID-19 Study Register (including Cochrane Central Register of Controlled Trials, MEDLINE, Embase, ClinicalTrials.gov, WHO ICTRP, medRxiv), Web of Science and WHO COVID-19 Global literature on COVID-19 to identify completed and ongoing studies up to 1 February 2022. Selection criteria We included RCTs that compared fluvoxamine in addition to standard care (also including no intervention), with standard care (alone or with placebo), or any other active pharmacological comparator with proven efficacy in clinical trials for the treatment of people with confirmed COVID-19, irrespective of disease severity, in both inpatients and outpatients. Co-interventions needed to be the same in both study arms. We excluded studies comparing fluvoxamine to other pharmacological interventions with unproven efficacy. Data collection and analysis We assessed risk of bias of primary outcomes using the Cochrane Risk of Bias 2 tool for RCTs. We used GRADE to rate the certainty of evidence to treat people with asymptomatic to severe COVID-19 for the primary outcomes including mortality, clinical deterioration, clinical improvement, quality of life, serious adverse events, adverse events of any grade, and suicide or suicide attempt. Main results We identified two completed studies with a total of 1649 symptomatic participants. One study was conducted in the USA (study with 152 participants, 80 and 72 participants per study arm) and the other study in BraziE (study with 1497 high-risk participants for progression to severe disease, 741 and 756 participants per study arm) among outpatients with mild COVID-19. Both studies were double-blind, placebo controlled trials in which participants were prescribed 100 mg fluvoxamine two or three times daily for a maximum of 15 days. We identified five ongoing studies and two studies awaiting classification (due to translation issues, and due to missing published data). We found no published studies comparing fluvoxamine to other pharmacologica interventions of proven efficacy. We assessed both included studies to have an overall high risk of bias. Fluvoxamine for the treatment of COVID-19 in inpatients We did not identify any completed studies of inpatients. Fluvoxamine for the treatment of COVID-19 in outpatients Fluvoxamine in addition to standard care may slightly reduce all-cause mortality at day 28 (RR 0.69, 95% Cl 0.38 to 1.27; risk difference (RD) 9 per 1000; 2 studies, 1649 participants; low-certainty evidence), and may reduce clinical deterioration defined as all-cause hospital admission or death before hospital admission (RR 0.55, 95% CI 0.16 to 1.89; RD 57 per 1000; 2 studies, 1649 participants; low-certainty evidence). We are very uncertain regarding the effect of fluvoxamine on serious adverse events (RR 0.56, 95% CI 0.15 to 2.03; RD 54 per 1000; 2 studies, 1649 participants; very low-certainty evidence) or adverse events of any grade (RR 1.06, 95% CI 0.82 to 1.37; RD 7 per 1000; 2 studies, 1649 participants; very low-certainty evidence). Neither of the studies reported on symptom resolution (clinical improvement), quality of life or suicide/suicide attempt. Authors' conclusions Based on a low-certainty evidence, fluvoxamine may slightly reduce all-cause mortality at day 28, and may reduce the risk of admission to hospital or death in outpatients with mild COVID-19. However, we are very uncertain regarding the effect of fluvoxamine on serious adverse events, or any adverse events. In accordance with the living approach of this review, we will continually update our search and include eligible trials as they arise, to complete any gaps in the evidence

Thiopental induces eEF2 phosphorylation.

<p>SK-N-SH cells were treated with 10 µM –2 mM thiopental for 6 h (A) or with 0.5 mM thiopental for 10 min to 12 h (B) and analyzed by immunoblotting with an anti-human phospho-eEF2 threonine 56 antibody (upper blots) or an eEF2 antibody that detects endogenous levels of eEF2 independently of phosphorylation (lower blots). Data are representative of four independent experiments.</p

Inhibitors of protein synthesis preserve intracellular ATP-content during oxygen deprivation.

<p>Human neuronal SK-N-SH cells were cultured in an oxygen-free atmosphere for 12–72 h in the presence of 5 µg/ml cycloheximide (closed triangles), 0.1 mM thiopental (closed rhombi), 0.5 mM thiopental (asterisks), or left untreated (closed squares). Cells, cultured in a normoxic atmosphere (open squares) served as a control. ATP content of cells was measured in lysates by an ATP-driven luciferase assay. Determined relative light units (RLU) were normalized to protein content and represent the means ± standard deviations of three independent experiments. Experimental groups were statistically evaluated by performing two-way ANOVA followed by the Bonferroni’s <i>post hoc</i> test. Statistical differences of oxygen deprived SK-N-SH cells (closed squares) compared to oxygen-deprived cells treated with 5 µg/ml cycloheximide (^###, p<0.001), 0.5 mM thiopental (^{<>\raster(60%)="rg3"<>}, p<0.05; ^{<>\raster(60%)="rg3"<><>\raster(60%)="rg3"<>}, p<0.01), 0.1 mM thiopental (^¥¥, p<0.01), or untreated control cells (^§§, p<0.01; ^§§§, p<0.001) are shown.</p

Inhibitors of protein synthesis reduce hypoxic neuronal damage.

<p>Cellular damage in human neuronal SK-N-SH cells was induced by oxygen deprivation (closed symbols) in an atmosphere containing 5% CO₂, 95% N₂ for 0–72 h and determined by an LDH release assay. Control cells were cultured in 5% CO₂, 21% O₂, and 74% N₂ (open symbols). In (A), cellular damage was measured in the presence (squares) or absence (rhombi) of fetal calf serum. In (B), 5 µg/ml cycloheximide (closed triangles) or 2 µg/ml actinomycin D (closed rhombi) were added to the cells in serum containing growth medium (squares). In (C), 0.1 mM thiopental (closed circles) or 0.5 mM thiopental (closed triangles) were added to the cells in serum containing growth medium (squares). Values represent the mean ± standard deviations of four separate experiments. Experimental groups were statistically analyzed by performing two-way ANOVA followed by the Bonferroni’s <i>post hoc</i> test. Statistically significant differences within groups shown for (A) are: serum treated oxygen deprived SK-N-SH cells versus serum treated normoxic control cells (***, p<0.001). Statistically significant differences within serum treated groups shown for (B) are: normoxic control cells versus oxygen deprived SK-N-SH cells (***, p<0.001); and oxygen deprived SK-N-SH cells versus oxygen-deprived cells treated with 2 µg/ml actinomycin D (^§§§, p<0.001) or versus oxygen-deprived cells treated with 5 µg/ml cycloheximide (^###, p<0.001). Statistically significant differences within serum treated groups shown for (C) are: normoxic control cells versus oxygen deprived SK-N-SH cells (***, p<0.001), versus oxygen-deprived cells treated with 0.1 mM thiopental (^{<>\raster(60%)="rg1"<>}, p<0.05; ^{<>\raster(60%)="rg1"<><>\raster(60%)="rg1"<><>\raster(60%)="rg1"<>}, p<0.001) or versus oxygen-deprived cells treated with 0.5 mM thiopental (^{<>\raster(60%)="rg2"<><>\raster(60%)="rg2"<><>\raster(60%)="rg2"<>}, p<0.001); and oxygen deprived SK-N-SH cells versus oxygen-deprived cells treated with 0.1 mM thiopental (^{<>\raster(60%)="rg3"<><>\raster(60%)="rg3"<><>\raster(60%)="rg3"<>}, p<0.001) or versus oxygen-deprived cells treated with 0.5 mM thiopental (^¥¥¥, p<0.001).</p

Thiopental inhibits protein synthesis, ameliorates hypoxic damage, and maintains energy balance during oxygen deprivation in primary cortical neurons.

<p>In (A), cortical neurons were treated with 10 µM −2 mM thiopental for 30 min and analyzed for phosphorylation of eEF2 and AMPK by immunoblotting. In (B), cortical neurons were left untreated or were exposed to 0.1–2 mM thiopental for 6 h and then pulse labeled with 200 µCi of [³⁵S]methionine for an additional 2 h. Cellular lysates were separated by 10% SDS-PAGE and the amounts of newly synthesized proteins were detected by autoradiography on dried electrophoresis gels. In (C/D), cortical neurons were exposed to hypoxia for 48 h in the presence or absence of 0.5 mM thiopental. Cellular damage was determined by an LDH-release assay (C). The relative intracellular ATP-content was measured by an ATP-driven luciferase assay (D). Values represent the mean ± standard deviations of three independent experiments. Statistical evaluation of experimental groups was performed by one-way ANOVA followed by the Bonferroni’s <i>post hoc</i> test. The statistically significant difference of oxygen-deprived cortical neurons in the presence or absence of thiopental is shown (***, p<0.001).</p